Biological sample storage device and method for biological sample contamination testing

ABSTRACT

The present invention is directed to a method for screening blood and blood products that utilizes a specialized blood and blood products storage device useful for storing and testing blood or blood products comprising a container having at least one compartment with one or more sections for storing and testing the blood or blood products for a target molecule. The method utilizes a homogeneous testing system that employs ribozymes, DNAzymes or catalytic antibodies.

BACKGROUND OF THE INVENTION

[0001] This invention relates generally to the field of screeningbiological samples and specimens for contamination by a targetedmolecule that is indicative of the presence of a pathogen, infectiousagent or contaminating agent. More specifically, the invention isdirected to a method of screening blood and blood products that utilizesa specialized blood and blood products storage device useful for bothstoring and testing blood and blood products. The storage devicecomprises a container having at least one compartment with one or moresections for storing and testing the blood or blood products for atarget molecule that is indicative of the presence of a pathogen, suchas a bacterium, virus, parasite, prion or other pathogenic agent.

[0002] Currently, blood banks use several individual assays to test forthe presence of multiple agents or molecules associated with orindicative of a disease or condition. Exemplary agents or moleculesinclude anti-HIV I/II antibodies, anti-HCV antibodies, HBV surfaceantigen, anti-HBC antibodies, liver enzyme (ALT), syphilis and HIV P24antigen. Additionally, infectious agents such as pathogenic bacteria andfungi, prions and protozoa, including the causative agents of small pox,malaria, West Nile disease, Chagas disease, and variant Creutzfel Jacobdisease (vCJD) may be detected. Additionally the TMA Procleix system iscurrently used to test for HCV and HIV-1 viral RNA molecules.

[0003] The current method involves collecting blood directly into ablood collection device via a collection needle and duct, and collectinga separate fraction of blood for testing/screening for various diseases.In addition to concerns about contamination of the blood by agents ormolecules that indicate an infection in the blood donor, a concern alsoexists that the techniques used for blood collection may causecontamination of the collected blood. For example, when blood iscollected via a needle, there may be bacterial contamination from skinor a hair follicle caught in the needle that could get into thecollected blood, as well as bacterial contamination inadvertentlyintroduced from other aspects of collecting and handling the blood andblood products. Contaminating bacteria replicate within blood and cancause adverse effects in recipients. For example, contaminating bacteriacan produce high levels of endotoxins and other pathogenic componentsthat cause severe health damage, including death. This problem isparticularly keen for platelets, a blood product separated from thewhole blood that must be stored at room temperature until use.Presently, platelets only can be stored up to 5 days, and must bediscarded if not used during that time. If, however, the platelets couldbe tested quickly, simply and accurately for contamination immediatelyprior to use, the length of storage time could be increased.

[0004] Currently, there is an issue at blood banks and hospitals overthe difficulty of detecting and evaluating bacterial and other pathogenlevels in blood because the pathogens must replicate to reach thresholdlevels for detection. Detection at Day 0 of collection, therefore, isnot a sufficient measure for determining that blood is free of bacteriaand other pathogens. Also, some forms of bacteria take longer toreplicate and to become detectable, particularly if the blood or bloodproducts are stored at approximately 4° C.

[0005] Under current practices, blood banks must make a difficultdecision as to what is an acceptable level of bacterial contaminationfor a blood product to be considered safe for release to a patient.Thus, the timing, simplicity and sensitivity of screening tests is animportant consideration. Testing done at the time of collection, Day 0,or shortly thereafter needs to be extremely sensitive because it islikely that the number of bacteria present at Day 0 is very low. Anextremely sensitive assay may result in a significant number of falsepositives, raising serious regulatory issues because the test should notresult in every unit of blood product testing positive. A furtherconsideration is that screening tests must be performed at the bloodbank before sending the sample to a hospital, or alternately at thehospital, immediately prior to use. The screening tests therefore mustbe rapid and fairly simple, such that they may be performed by medicaltechnicians.

SUMMARY OF THE INVENTION

[0006] The present invention provides a blood or blood products storagedevice for storing and testing blood or blood products that combines aunique configuration with a “real-time test” that includes state of theart diagnostic detection technology providing a visual signal detectionof pathogen contamination, particularly bacterial, viral and parasiticcontamination. More specifically, the claimed device comprises acontainer having at least one compartment with one or more sections fortesting biological samples, particularly blood and blood products, forcontamination by a pathogenic agent or a product of a pathogenic agent,such as a toxin. The invention also provides a method of using thedevice for testing samples for the presence of the pathogenic agent orproduct. The container design provides the opportunity for collecting,storing and testing, all in the claimed device. In one embodiment, thespecialized multi-sectioned compartmentalized device is self-containedfor the storage and testing of the blood or blood product withoutopening the container and exposing the sample to additional risk ofcontamination. Although the device and method preferably are designed todetect bacterial contamination in the blood, i.e., bacteria or abacterial product such as toxins, the device and method can be used todetect the presence of any pathogen in the blood or blood product, suchas viruses, viral components or products, parasites, parasiticcomponents or products or other detectable components of the indicativeof the presence of pathogenic agents.

[0007] The present device provides a self-contained storage and testingcontainer that facilitates a fast and easy way to test blood or bloodproducts throughout its shelf-life, while conserving collected blood andnot exposing it to manipulations that may cause further contamination.The configuration of test reagents used in the device allows them toremain separated from the blood or blood product until the moment oftesting.

[0008] An advantage of the invention is the concept of “real timetesting,” in which the presence of bacteria or any pathogen or theirproducts of interest in the blood or blood product can be tested ormonitored in real time, on an ongoing basis up to the actual time ofrelease of the blood or blood product to a patient. A further advantageof the invention is that the testing assay is a homogenous assay,meaning that there are no separate washing steps involved. The testreagents are mixed and the signals are generated and detected without aneed for any further manipulations. Any known homogeneous signaldetection assays that can be utilized as the signal-detection testingprocedure with the disclosed device. Exemplary signal detection assaysemploy ribozyme, DNAzyme or catalytic antibody technology. Anotheremploys B-cells engineered to express aeqorin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in andconstitute a part of the specification, illustrate presently preferredembodiments of the invention. Together with the general descriptiongiven above and the detailed description given below, the drawings serveto explain principles of the invention.

[0010]FIG. 1 shows one embodiment of a biological sample storage deviceof the present invention.

[0011]FIG. 2 shows one embodiment of the testing reagents, utilizing asa solid support a biochip, and the relationship and interaction of thetarget molecule, the ribozyme and the RNA reporter gene sequences.

[0012]FIG. 3 shows one embodiment of the interaction of the targetmolecule, the ribozyme and the RNA reporter gene sequences when theribozyme designated as a “Halfzyme

” and the reporter gene are immobilized on a solid support.

[0013]FIG. 4 shows another embodiment of the interaction of the targetmolecule, the ribozyme and the RNA reporter gene sequences when theribozyme is in a liquid phase and the reporter gene is immobilized on asolid support, such as a biochip or beads.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[0014] The reference throughout this application alternately to blood orblood products is intended to encompass either of these biologicalsamples. Further, the word “a” or “an” is intended to encompass one ormore recited elements.

[0015] The present invention is directed to a biological sample storagedevice for storing and testing blood or blood products. The devicecomprises a container for receiving and storing blood or blood products,and at least one compartment for testing the blood or blood products.The compartment comprises at least a first section for holding a portionof the blood or blood products, and optionally testing the portion ofthe blood or blood products. The first section of the device is arrangedcontiguous to the container so that the blood products can flow from thecontainer to the first section. The first section further is designedsuch that the first section can be sealed from the container, so thatthe blood products sealed into the first section can be used fortesting. The device further comprises at least one additional sectionfor testing the portion of the blood or blood products held in the firstsection, the additional section being arranged in sealed contact withanother portion of the first section, different from a portion of thefirst section in contact with the container. The additional sectioncomprises a pressure sensitive seal between the first section and theadditional section that can broken by the application of pressure, suchthat breaking the seal in the additional section allows mixing of thecontents of the first and additional sections. The container of thedevice comprises a plurality of compartments. In one embodiment, each ofthe compartments is arranged as a protruding element from the container.

[0016] In another embodiment, the device comprises a first section andat least one additional section comprising a second and a third section.The second section is arranged in sealed contact with another portion ofthe first section, different from a portion of the first section incontact with the container. The third section is arranged in sealedcontact with another portion of the second section, different from aportion of the second section in sealed contact with the first section.Further, the second and third sections comprise pressure sensitive sealsthat can broken by the application of pressure such that breaking theseal in the second section allows mixing of the contents of the firstand second sections and breaking of the seal in the third section allowstransfer of the mixed contents of the first and second sections into thethird sections. The second section contains a buffer, wherein the bufferis a lysis buffer or an isotonic buffer, and the third section containstest reagents for testing the transferred mixed contents from the firstand second sections, wherein these test reagents are a catalyticmolecule and a reporter sequence. In one embodiment, the catalyticmolecule is an inactivated ribozyme, a DNAzyme or a catalytic antibody.In a further embodiment, the test reagents are an inactivated ribozymeand an RNA reporter sequence. At least one of the test reagents, thecatalytic molecule or the reporter sequence is immobilized to a solidsupport or at least one of the test reagents, the catalytic molecule orreport sequence is in a lyophilized form. In one embodiment, the bloodproducts tested in the device comprise blood platelets.

[0017] Any number of solid support systems can be used for immobilizingtest reagents. For example, the support may be a biochip, a bead, a gel,a microparticle, a membrane, a slide, a plate, a pellet, a disk, acapillary, a hollow fiber, a needle, a solid fiber or another formcapable of providing a substrate for attachment of the test reagent(s).The support system may comprise a wide variety of compositions,including glass, plastic, silicon, alkanethiolate-derivatized gold,cellulose, low crosslinked and high crosslinked polystyrene, silica gel,polyamide, and the like.

[0018] In one embodiment, the blood or blood products storage deviceaccommodates, in its compartment for testing the blood or bloodproducts, biosensors capable of supporting a “self-performing” screeningassay. For example, engineered B-cells with surface antibodies specificfor target agents or molecules may be attached to a biochip. Theengineered B-cells express cytosolic aequorin, a calcium-sensitivebioluminescent protein from Aequoria Victoria jellyfish. Once a targetantigen binds to the B-cell, it will trigger an elevated intracellularcalcium concentration, which will illuminate aequorin. See, e.g., Rideret al., Science, 301: 213-215 (2003). Such a “self-performing” screeningassay is particularly advantageous because it also allows for continuousmonitoring of a blood sample by visual inspection.

[0019] The invention is further directed to a method of testing blood orblood product for a target molecule indicative of contamination in theblood or blood product. The method comprises (a) providing a sample of ablood product in a compartment of the storage device for storing andtesting blood or blood products, which storage device comprises acontainer for receiving and storing blood or blood products and at leastone compartment for testing the blood or blood products, wherein thecompartment comprises at least a first section for holding a portion ofthe blood or blood products, and optionally testing the portion of theblood or blood products; (b) contacting the blood product in thecompartment with a lysing buffer; (c) releasing the target molecule fromthe cells and protein in the blood product; and (d) detecting thepresence of the target molecule. In one embodiment the target moleculeis a 16S ribosomal RNA or a nucleic acid associated with a pathogen. Thedetecting step of the method may employ test reagents comprising acatalytic molecule and a reporter sequence, wherein the catalyticmolecule is an inactivated ribozyme, a DNAzyme or a catalytic antibody.In a further embodiment, the test reagents are an inactivated ribozymeand an RNA reporter sequence. In this embodiment of the method, theinactivated ribozyme binds to the target molecule under sufficientconditions to activate the ribozyme that in turn cleaves the RNAreporter sequence and releases a detectable sequence.

[0020] In another embodiment of the method, the target molecule in thedescribed method is a protein that is associated with or indicative of apathogen. In the alternate method of testing a blood or blood productfor these target proteins, which generally are cell surface proteins orsecreted proteins of pathogens, such as bacteria, viruses, parasites andother pathogenic agents, the buffer is an isotonic buffer that dilutesthe sample and does not lyse the cells or degrade the proteins. Themethod is performed in the same way as in the method of detecting thenucleic acid, but utilizes an isotonic buffer that does not lyse thecells or pathogen. The detecting step of the method employs testreagents comprising a catalytic molecule and a reporter sequence,wherein the catalytic molecule is an inactivated ribozyme, a DNAzyme ora catalytic antibody. In a further embodiment, the test reagents are aninactivated ribozyme and an RNA reporter sequence. In this embodiment ofthe method, the inactivated ribozyme binds to the target molecule undersufficient conditions to activate the ribozyme that in turn cleaves theRNA reporter sequence and releases a detectable sequence.

[0021] As shown in one aspect of the invention in FIG. 1, the presentinvention provides a storage device (10) for collection, storage andtesting of blood or blood products comprising at least one compartment(20), preferably a plurality of compartments, each with at least twosections (for examples, the sections (30), (40) or (50)), with onesection (30) for holding a portion of the blood product that will betested and at least one additional section (for example, section (50))for testing the portion of the blood or blood products present in thefirst section (30). Each of the compartments (20) is preferably arrangedas a protruding element from the container (15) but a configuration ofthe compartment(s) within the device may be such that they are formedwithin the device and do not protrude outwardly from the device wall(16). Each of the compartments (20) of the device is arrangedcontiguously to the container (15) so that the blood products can flowfrom the container (15) to the first section (30) of the compartment(20). The first section (30) is designed such that it can be sealed fromthe container (15) so that the blood products sealed into the firstsection (30) can be used for testing but separated from the remainingblood product in the container to maintain the integrity of the product.Heat sealing is one method for sealing off the first section (30) fromthe container. Each of the compartments (20) further comprise at leastone additional section (for e.g., section (50)) which contains the testreagents necessary for testing the blood product for contamination.Preferably this additional section comprises a second (40) and a thirdsection (50), the second section (40) being arranged in sealed contactwith another portion of the first section (30), different from a portionof the first section (30) in contact with the container (15), andwherein the third section being arranged in sealed contact with anotherportion of the second section (40), different from a portion of thesecond section (40) in sealed contact with the first section (30).Further, the second and third sections (40) and (50) include pressuresensitive seals that can be broken by the application of pressure, suchthat breaking the seal in the second section (40) allows mixing of thecontents of the first and second sections (30) and (40), and breakingthe seal in the third section (50) allows transfer of the mixed contentsof the first and second sections (30) and (40) into the third section(50).

[0022] The blood products storage device (10) preferably is a bagcomposed of a flexible material, such as polyvinylchloride (PVC) orother similar type of material that will accommodate the design of thedevice and not adversely affect the blood product. For example,thermoplastic polyolefins, such as polyethylene, polypropylene,polybutalene and copolymers composed of such materials can be used.These can be made by Ziegler-Natta, metallocene or free-radicalcatalysts. Polyolefin blends also can be used in which polybutadiene andpolyisoprene are mixed in, as well as ethylene co-polymers (EVA=ethylenevinyl acetate and EMA=ethylene methyl acrylate). Different nylonmaterials also may be used to prepare the device (10).

[0023] The container (15) includes at least one and preferably aplurality of compartments (20), each comprising at least two sectionsbut more preferably three sections. In one embodiment, each of thecompartments (20) of the container (15) comprises three sections (30),(40) and (50), however, the present invention may be performed usingcompartments with more or less sections than three if the selectedtesting method requires more or less test reagents and manipulativesteps.

[0024] In all embodiments, the first section (30) of a compartment (20)is contiguous with the container so that it is in open connection to thecontainer (15) and contains the same blood product as is in thecontainer (15) prior to testing. Immediately prior to testing, thecontainer (15) is agitated to ensure that the blood product in the firstsection (30) of a compartment (20) is representative of the bloodproduct in the container (15). Then, this first section (30) of thecompartment (20) is permanently sealed off from the container (15) byforming a seal between the container (15) and the first section (30)forming seal #1 (32) as depicted in FIG. 1. The second section (40)which is sealed off from the first section (30) and third section (50)via pressure sensitive seals (42) and (52), contains a buffer, in oneembodiment a lysis buffer for lysing and degrading the cells, bacteria,any cellular or bacterial associated proteins and proteins present inthe blood product. When the sample in the first section (30) of thecompartment (20) is ready to test, the pressure sensitive seal, Seal #2(42), is broken with applied pressure, and the blood product sample heldin the first section (30) mixes with the lysing buffer. After asufficient period of time to achieve lysing of the blood product sample,seal #3 (52) is broken, allowing the contents of the first and secondsections (30) and (40) to flow into the third section (50), whichcontains the test reagents required to perform the test to detect thepresence of pathogen. In one embodiment, there are two test reagents inthe third section (50), which may both be bound to a solid support, suchas a biochip or bead, or lyophilized in the third section (50) so thatthey are maintained in a dry environment until use when they aresolubilized by the sample/buffer mixture. When the reagents are bound toa solid support, they remain bound during the testing; however, if oneor both of the test reagents are lyophilized in the third section (50)of the compartment (20), then these reagents will be in the liquid phaseinside the compartment (20) when pressure sensitive seal #3 (52) betweenthe second and third sections (40) and (50) is broken. The test reagentsin the third section (50) now mix with the sample/buffer mixture and thelevel of contamination can then be detected visually after completion ofsignal activation in the compartment (20). This test generally takesabout an hour and can be repeated for each of the compartments (20) onsubsequent days depending upon the number of compartments (20) presentin the container (15). Any number of compartments (20) can be present inthe container (15) and the device can be designed to contain a suitablenumber of compartments (20) for the particular use of the device.However, it is most likely that six compartments (20) will be present inthe container for testing from Days 2 to 7 as shown in FIG. 1. Thus, thedevice's contamination levels can be monitored everyday for a week orevery few days to extend the testing period.

[0025] This test can be administered by hospital staff one to two hoursbefore a transfusion to a patient, as the test requires only sealingseal #1 (32) and breaking seals #2 (42) and #3 (52) and reading thedetection signals. Other advantages of this design are based on theoptimization of the test itself.

[0026] Also the dry environment of the third compartment (50) assuresstabilization of the test reagents involved in the detection methoduntil the moment of testing. Any suitable test reagents can be employedas long as they provide the ability to detect the presence of thetargeted molecule indicative of a contaminating pathogen. In oneembodiment, the test reagents are allosteric enzymes and reporter genesequences that can be cleaved by the allosteric enzymes and located inthe third section (50) of each of the compartments.

[0027] An allosteric enzyme useful in the present invention is acatalytically inactive ribozyme that has a specific binding constant fora known target molecule that may be present in the blood product. Thetarget molecule contains a sequence that activates the ribozyme whichthen cleaves a specific cleavage site in the RNA reporter sequence.Similarly, DNAzymes can be substituted for the ribozymes; however, inthe discussions of the test reagents useful in this disclosure,ribozymes are used when DNAzymes could alternatively be used.

[0028] For detection of bacterial contamination, the preferred targetmolecule of the ribozyme is conserved bacteria-specific ribosomal RNA(rRNA), such as 16S rRNA, which detects the presence of bacteria in theblood product. In addition to targeting the specific sequences of 16SrRNA that indicates the presence of bacteria, specific ribozymes alsocan recognize sequences in the 16S rRNA to detect specific bacterialtaxonomic family/genus/species to which the contaminating bacteriabelong. For example, a first test is performed in one of thecompartments in which the ribozyme targets the conserved 16S rRNA todetect the presence of the bacteria. Then, a plurality of compartmentseach containing a specific ribozyme that targets specific 16S rRNA isused to detect the genus and species, and possibly strain of acontaminating bacteria. See U.S. Pat. No. 5,679,520 for use of 16S rRNAto detect particular species of bacteria. This type of testing is usefulbecause not all bacteria impose the same degree of health risk at agiven level of contamination. Such a panel of further tests forbacterial species provide more information regarding whether the levelof contamination with a particularly identified type of bacteria isacceptable or not in the blood product.

[0029] The bacteria that the device and test reagents are designed totest for are those that have contaminated the blood during collectionand handling or bacteria that have infected the blood of the donor. Thebacteria may be Escherichia coli, Salmonella species, Campylobacterspecies, Listeria species, Enterococcus species, Neisseria gonorrhoeae,Enterobacter cloacae, Pseudomonas species, Proteus mirabilis,Legionella, Mycobacterium species, Chlamydia species, other types ofbacteria, protozoa and fungi.

[0030] The target molecule may also be nucleic acids, both RNA and DNAfrom viruses, such as HIV: long term repeated region (LTR), HBV: preSand S regions, HCV: 5′ untranslated region (UTR), particularly regionsof these viruses known to be indicative of the presence of theseviruses. Additionally, the target molecule may be nucleic acids fromparasites, such as Plasmodium falciparum, Trypanosoma cruzi, Trypanosomabrucei, Babesia microti and Leishmania tropica.

[0031] When nucleic acid is the target molecule, the lysis step ensuresthat the pathogenic-specific RNA sequences are properly exposed to allowfor easy access for binding to and activation of the allostericribozyme. This is important because the allosteric ribozyme or DNAzymegenerally can only recognize a range of 10-28 nucleotide sequences,disregarding secondary or tertiary structure as well. The need for anexposed linear epitope is satisfied by lysing the pathogen's cells. Aribozyme useful in the present invention is a ribozyme that has beendesigned to possess a specific binding affinity to a RNA target moleculeto ensure binding and to possess sequences that cleave a designated RNAreporter sequence. Cruachem, Ltd. is a commercial source for thesereporter molecules and provides services to design specific signalprobes with the desired fluorescent dyes. This company is also a sourceof specifically designed ribozymes for specific assays. In oneembodiment, the allosteric ribozyme is a catalytically inactiveribozyme, such as a “Halfzyme

,” a product of Ribozyme Pharmaceutical, Inc., Boulder Colo., (“RPI”),which is engineered so that the target molecule supplies the structuralelements; i.e., the missing sequences required for ribozyme catalysis.

[0032] The test reagents, e.g., the allosteric ribozymes and the RNAreporter sequences, are sensitive to the pathogen load in the bloodproduct. In the case of bacterial detection, with the bacterial specific16S rRNA sequence exposed in the lysate, the detection system utilizessequence-specific binding and activation of enzymes, that cascade to avisible signal. Once the bacterial load reaches a certain level orconcentration, a detectable signal is activated, which involves thecapture of bacteria-specific sequences, through the use of technology ofspecifically designed allosteric enzymes. It is believed that a singlebacterium present in the blood product is equivalent to 10³-10⁴ copiesof 16S rRNA. A single bacterium is equivalent to one colony forming unit(CFU) of a specific bacteria, which is defined as a single bacteriumthat is able to clone itself into an entire colony of identical cells.Therefore, 10 CFUs would result in approximately 10⁴-10⁵ 16S rRNA copiespresent in the lysis sample solution which provides sufficient analytesfor a very sensitive assay. The 16S rRNA is the target of the assay.

[0033] Ribozymes can be designed to have specified binding constantswith the desired target sequence. Depending on the concentration of thetarget molecule in the blood product, the allosteric enzyme/ribozyme isactivated to recognize and cleave an RNA reporter sequence with aquenched signal attached to the solid surface or carrier or in solutionin the lysed sample/buffer mixture. The ribozyme will not cleave the RNAreporter sequence if the concentration of the target 16S rRNA sequencesis not high enough. Since the ribozyme is designed to have specifiedbinding constants, the activation of the ribozyme is dependent on theconcentration of the 16S rRNA target sequences present in the blood. Theribozymes that recognize target RNA can be prepared using known methods,as disclosed in RPI patents, such as U.S. Pat. Nos. 6,482,932;5,496,698, and Vaish et al., Nature Biotechnology, Vol. 20, 810-815(August 2002), all of which are incorporated by reference in theirentirety.

[0034] The homogenous assay used in the present invention can utilizeribozymes, DNAzymes or catalytic antibodies as the signal-detectiontechnology. The present invention is described in the context ofutilizing ribozymes but any technology can be used with the disclosedblood storage device that provides a homogeneous assay and thegeneration of a detectable signal. The basic concept of the use ofDNAzymes and catalytic antibodies are similar to the ribozymes describedherein. Regulators or effectors bind to DNAzyme (instead of RNAzyme inthe case of ribozyme) and activate the RNA-cleaving activity of theDNAzyme. DNAzymes may have better stability and easier to synthesizethan ribozymes. Both DNAzymes and catalytic antibodies are knowntechnologies that have been described in the scientific literature. Thefollowing publications describe this technology and the preparation andassays using these reagents are known in the prior art. For example, (1)A General Strategy for effectors-mediated Control of RNA-cleavingRibozymes and DNA Enzymes, D. Y. Wang, B. H. Y. Lai and D. Sen, J. Mol.Biol. (2002), 318, 33-43; (2) A general approach for the use ofoligonucleotide effectors to regulate the catalysis of RNA-cleavingribozymes and DNAzymes. D. Y. Wang, B. H. Y. Lai, A. R. Feldman, and D.Sen, Nuc. Acid Res., 2002, 30, 1735-42; (3) Cleaving DNA with DNA. N.Carmi, S. R. Balkhi, and R. R. Breaker. Proc. Natl. Acad. Sci, 1998, 95,2233-2237; (4) A circular RNA-DNA enzyme obtained by in vitro selection.X D. Kong, S Z. Zhu, X J Guo, X P. Wang, H Y Zhang, and J. Zhang.Biochem Biophys Res Commun., 2002, 292, 1111-5; (5) Catalytic DNA: anoval tool for gene suppression. M J Cairns, E G Saravolac and L Q Sun.,Curr Drug Targets 2002, 3, 269-79; AND (6) Development of a GeneticSelection for Catalytic Antibodies. J. Gildersleeve, J. Janes, H.Ulrich, P. Yang, C. Barbas, and P. G. Schultz. Bioorganic & MedicinalChemistry Letters 2002, 12 1691-94, which are all herein incorporated byreference.

[0035] Once the RNA reporter sequence is cleaved, the quenched signal isreleased, and detection is then complete. FIG. 2 provides an example ofthe test reaction that occurs, showing that the target 16S rRNA sequence(100) from a specific strains of bacteria bind or hybridize to aspecifically designed ribozyme (110) with a specified binding constantfor sequences in the targeted 16S rRNA. When the concentration of thetarget 16S rRNA (100) sequence is sufficient to activate the ribozyme(110), the ribozyme (110) will cleave the RNA reporter sequence (120),that in this embodiment is attached to a solid support, such as biochipor beads, (130) as carrier. The biochip can be made from any suitableknown materials, such as activated glass, plastic material, such aspolystyrene or nylon, and ceramic materials. The beads likewise can bemade from known materials, such as polystryrene. A detectable quenchedsignal (140) is released and photons are generated. This signal can bevisually detected as a fluorescent signal using appropriate ultraviolet(U.V.) analytical equipment, such as a U.V. reading box, a fluorescencespectrophotometer (Beckman DU640 scanning U.V.-vis or Hitachi, San Jose,Calif.—Model F4500) or a fluorimeter, such as the Jenway Model 6200Fluorimeter. A quantitative signal can be measured using a digitalcamera that scans U.V. light or fluorescence which are available fromKodak, BioRad or Alph Innotech, Inc. The compartment (20) can remainattached to the container (15) during the fluorescence signaldetermination or the compartment can be cleaved from the container (15)at the permanent Seal #1 (32) being careful not to damage the container(15) or the compartment (20).

[0036] In regard to the configuration shown in FIG. 2, it should benoted that only one terminal of the reporter gene, for example of abiotinylated molecule, need be attached to the solid support because thereporter gene and signal probe structure should have some mobility toallow the quencher molecule and reporter dye to split and diffuse awayfrom each other. Thus, in a configuration of the test reagents whereboth the ribozyme and the reporter sequence are immobilized on the solidsupport at a portion of these molecules and these reagents should beimmobilized so that the signal portion and the quencher portion of thereporter sequence are free to allow some mobility so that the signal canbe released.

[0037]FIGS. 3 and 4 provide more detailed disclosure of twoconfigurations of the test reagents in the third section in the presenceof the sample/buffer mixture used to detect specific bacterial 16S RNA.

[0038] In FIG. 3, the allosteric inactivated ribozyme and the reportergene are immobilized on the solid support. Once the 16S rRNA targetsequence binds to the ribozyme, it induces a conformational change andactivates the ribozyme. More specifically, approximately a 10 to 28nucleotide conserved sequence of 16S rRNA (conserved among all bacteriaor conserved among a species of bacteria) binds and incorporates itselfinto the ribozyme. In theory, these ribozymes are responsive to smallmolecule binding, which converts them to a secondary active stage. Theribozyme additionally contains a sequence that is capable of cleavingthe RNA reporter sequence that also is bound to the biochip. When theribozyme is activated by binding to the 16S rRNA, it then cleaves theRNA reporter sequence. The RNA reporter sequence is composed of a 5′fluorescent reporter dye, such as FAM (carboxyfluorescein) and a 3′quencher molecules, such as DABCYL (4-dimethylaminophenylazobenzoyl).Other reporter dyes that are available for use in the present inventioninclude TET and JOE. The RNA reporter sequence is designed to form astructure that brings into close proximity the 5′ and 3′ ends of theprobe, which quenches the fluorescent signal. Once the ribozyme cuts thecleavage site in the RNA reporter sequence, the FAM dye and the DABCYLseparate and give off a strong fluorescent signal as an electron jumpsdue to the split of these two molecules.

[0039]FIG. 4 provides an alternate configuration of test reagents insection 3, in which the ribozyme is lyophilized and placed in the thirdsection (50) of the compartment (20) or it is introduced through thewall of the third section in a liquid form via a syringe. The RNAreporter sequence is bound to the biochip. The 16S rRNA sequence bindsto complementary sequences in the catalytic site of the ribozyme andactivates it, forming a conformational change, which in turn cleaves theRNA reporter sequence at the cleavage site, which the ribozymespecifically recognizes. The signal is generated as described for FIG. 3above. The fluorescent signal results as an electron jumps from thedonor to the acceptor and the quencher molecule (DABCYL) and dye split(FAM) apart to release the quenched signal.

[0040] A further test reagent configuration provides the ribozyme boundto the biochip and the RNA reporter gene in liquid form that is eitherlyophilized and placed inside the third section (50) of the compartment(20) or introduced via a syringe into the third section (50) in liquidform.

[0041] A further test reagent configuration provides both the ribozymeand the RNA reporter sequence in a liquid, either as lyophilizedreagents in the third section or introduced via a syringe in solutioninto the third section (50). The ribozyme and the RNA reporter sequencecan be added simultaneously or sequentially with the simultaneousaddition preferred. Alternatively, if these two test reagents wereintroduced in liquid form via a syringe, it would not be necessary tohave a compartment with three sections. Only the first section (30)containing the blood sample and the second section (40) containing thebuffer would be necessary. In this embodiment, the ribozyme and RNAreporter molecule could be introduced into the second section (40) via asyringe. If the test reagents are not immobilized, it is useful toinclude a solubilizing agent in the buffer at about 1-2%, such as DMSO(dimethylsulfoxide) or other comparable agents.

[0042] Alternatively, the allosteric ribozyme recognizes a proteinrather than a nucleic acid. These ribozymes are known as “Allozymes,”also produced by RPI, and are activated by the molecular recognition ofa target protein. Vaish et al., Nature Biotechnology, Vol. 20, 810-815(August 2002) describe allosteric ribozymes that recognize proteins andis incorporated in its entirety by reference. The target proteins thatare recognized by the allozymes are cell surface proteins or secretedproteins of pathogens, such as bacteria, viruses, parasites and otherpathogenic agents. If Allozymes are employed, the blood product iscollected in the first section (30) of compartment (20) and sealed offat Seal #1 as discussed above. Pressure is placed on the pressuresensitive Seal #2 (42) and the blood product flows into the secondsection (40) which contains an isotonic buffer that dilutes the samplebut does not lyse the cells or degrade the proteins present. Aftersufficient mixing in the dilution buffer, the pressure sensitive Seal #3(52) is broken and the diluted blood sample flows into the third sectionin which the test reagents are immobilized or lyophilized and ready togo into solution. All of the various permutations of the combination,formulation and location of the test reagents applies to the detectionof pathogenic proteins in the sample.

[0043] The container (15) can be configured to contain compartments inwhich some contain the lysing buffer and some contain the dilutionbuffer. Alternatively, the compartments can contain two sections, onefor holding the blood sample to be tested and the other for holding thereagents. In this latter configuration, the buffer can be injected intothe compartment. In another embodiment, the compartment can have onesection for holding the blood sample. In this case, the appropriatebuffer and test reagents can be injected into the compartment and readfor signal release.

Experimental Section EXAMPLE 1

[0044] The claimed device in the form of a PVC bag with six compartmentsfor performing the contamination test is filled with blood or a bloodproduct, such as the platelet fraction, as shown in FIG. 1. Eachcompartment corresponds to Days 2 through 7, respectively. Eachcompartment is of sufficient size to test 1 ml of the collectedplatelets, which means that section 1 of each compartment holds a volumeof 1 ml. On Day 2, the bag is mixed and the platelet mixture flows intothe first compartment. Seal #1 is permanently sealed. Pressure is thenplaced on section 1 to break the pressure sensitive Seal #2 allowing theplatelets to flow into section 2 mixing with the lysis buffer containedin section 2. An effective lysis buffer is composed of a non-ionicdetergent, such as Triton X-102 (1-2%), protease enzymes (such asprotease K), Li chloride (1.0 M) and other protein denaturationreagents, such urea, guanidine HCl, EDTA (5 mM) or EGTA, and relatedinhibitors of nucleases; and related protein solubilizing reagents;Hepes (50 mM pH 7.5) and related buffering reagents. Any lysis buffer isintended to be useful in the present invention as long as it is capableof lysing cells, bacteria, and proteins and release the nucleic acidsfrom cellular and bacterial protein components to provide targets forribozyme activation without interference with the reporter gene sequenceand functions. Other buffers and components of a lysis buffer that canlyse cells and degrade proteins to release 16S rRNA target sequences areknown to persons skilled in the art. The buffer system for the ribozymeassay may include a combination of metal ions, such as Pb²⁺ and Nd³⁺(neodymium), as well as divalent ions, such as Mg²⁺ and Mn²⁺, foroptimizing ribozyme activity, i.e., cleavage of the target sequence). Itshould be noted that only one terminal of the reporter gene, for examplesuch as a biotinylated molecule, and that the reporter gene and signalprobe structure must have some mobility to allow the quencher moleculeand reporter dye to split and diffuse away from each other. Thus, in aconfiguration of the test reagents where both the ribozyme and thereporter sequence are immobilized on the solid support, these reagentsshould not be immobilized at all points of the molecules, and in fact,the signal molecule and the quencher should be free to allow somemobility so that the signal can be released.

[0045] After lysing the platelet sample for a sufficient period of time,approximately 10 to 20 minutes to obtain a relatively clear mixture, torelease the bacterial nucleic acid, the pressure sensitive seal #3 isbroken to allow the mixed contents of the first and second sections (30)and (40) to flow into the third section (50). If the numbers of bacteriahave reached a certain concentration level, the number of copies of the16S rRNA will be sufficient so that the allosteric enzyme is activatedto cut the RNA reporter sequence and a detectable signal is released.The binding of the allosteric enzyme to the target sequence isdetermined by specified binding constants, and is dependent on thetarget sequence concentration.

[0046] Generally, the hybridization temperature at which the ribozymebinds to the target molecule depends mainly on two factors: (1) thelength of the nucleic acid that is hybridized and (2) the saltconcentration within the buffer system. A temperature range of about45-60° C. is preferred. Known buffers that facilitate the hybridizationwithout denaturing and changing the hybridization profile of theribozymes is preferred and are known to persons skilled in the art.Ribozyme activation conditions are disclosed in several of the recitedpublications in this disclosure and can be utilized to perform thetesting method of the present invention. Also applying a heat lamp orheat block to the compartment may be helpful to accelerate the assay aswell as to increase its specificity.

[0047] The detectable sequence is fluorescent and can be visually readby an appropriate U.V. measuring device, such as a fluorescentspectrophotometer or U.V. light box. The total time for the assay isapproximately 1 to 2 hours; however, the assay time can be shortened byheating the compartment once the test reagents are added, such as byusing a U.V. light box that contains a heat lamp or a heating block.

[0048] The publications and patent documents cited herein areincorporated in their entirety by reference. Other embodiments of theinvention will be apparent to those skilled in the art from aconsideration of the specification and the practice of the inventiondisclosed herein.

EXAMPLE 2

[0049] This example demonstrates the use of a biochip coated withantibody to capture corresponding antigen in blood and detection of thetarget antigen by mass spectroscopy.

[0050] A biochip (Ciphergen PS20 Protein chip) was covalently bound to amouse monoclonal antibody against HIV P-24 core protein. The biochipthen was placed in a tube where 12 ml of human blood was circulated aflow rate of about 30 ml/min at 37° C. HIV P-24 antigen was spiked inthe blood at a concentration of 0-200 ng/ml. After 10 min ofcirculation, the chip was removed and the bound antigen was detected bymass spectroscopy (Ciphergen Protein Chip Reader).

[0051] The results showed that antigen can be detected at aconcentration of about 8 ng/ml in a 12 ml blood sample. FIG. 14 depictsa typical result.

EXAMPLE 3

[0052] This example demonstrates the use of a biochip coated withantigen to capture corresponding antibody in blood and detection of thetarget antibody by mass spectroscopy.

[0053] A biochip (Ciphergen PS20 Protein chip) was covalently bound toHIV P-24 core protein. The biochip then was placed in a tube where 12 mlof human blood was circulated at a flow rate of about 30 ml/min at 37°C. Mouse monoclonal antibody against HIV P-24 was spiked in blood at aconcentration of 0-20 ug/ml. After 10 min. of circulation, the chip wasremoved and the bound antibody was detected by mass spectroscopy(SELDI-MS from Ciphergen).

[0054] The results showed that antibody can be detected at aconcentration of about 1 ug/ml in a 12 ml blood sample.

We claim:
 1. A biological sample storage device for storing and testing blood or blood products, comprising: a container for receiving and storing blood or blood products; and at least one compartment for testing the blood or blood products, wherein said compartment comprises: at least a first section for holding a portion of the blood or blood products, and optionally for testing the portion of the blood or blood products.
 2. The device according to claim 1, wherein the first section is arranged contiguous to the container so that the blood products can flow from the container to the first section.
 3. The device according to claim 2, wherein the first section is designed such that the first section can be sealed from the container so that the blood products sealed into the first section can be used for testing.
 4. The device according to claim 2, further comprising at least one additional section for testing the portion of the blood or blood products held in the first section, said additional section being arranged in sealed contact with another portion of the first section, different from a portion of the first section in contact with the container.
 5. The device according to claim 4, wherein the additional section comprises a pressure sensitive seal between the first section and the additional section that can broken by the application of pressure, such that breaking the seal in the additional section allows mixing of the contents of the first and additional sections.
 6. The device according to claim 1, wherein the container comprises a plurality of compartments.
 7. The device according to claim 1, wherein each of the compartments is arranged as a protruding element from the container.
 8. The device according to claim 7, wherein the first section is arranged contiguous to the container so that the blood products can flow from the container to the first section.
 9. The device according to claim 8, wherein the first section is designed such that the first section can be sealed from the container so that the blood products sealed into the first section can be used for testing.
 10. The device according to claim 4, wherein the at least one additional section comprises a second and a third section, the second section being arranged in sealed contact with another portion of the first section, different from a portion of the first section in contact with the container, and wherein the third section being arranged in sealed contact with another portion of the second section, different from a portion of the second section in sealed contact with the first section.
 11. The device according to claim 10, wherein the second and third sections comprise pressure sensitive seals that can broken by the application of pressure such that breaking the seal in the second section allows mixing of the contents of the first and second sections and breaking of the seal in the third section allows transfer of the mixed contents of the first and second sections into the third sections.
 12. The device according to claim 11, wherein the second section contains a buffer, wherein said buffer is a lysis buffer or an isotonic buffer.
 13. The device according to claim 11, wherein the third section contains test reagents for testing the transferred mixed contents from the first and second sections.
 14. The device according to claim 13, wherein the test reagents are a catalytic molecule and a reporter sequence.
 15. The device according to claim 14, wherein said catalytic molecule is an inactivated ribozyme, a DNAzyme or a catalytic antibody.
 16. The device according to claim 14, wherein the test reagents are an inactivated ribozyme and an RNA reporter sequence.
 17. The device according to claim 14, wherein at least one of the catalytic molecule and reporter sequence is immobilized to a solid support.
 18. The device according to claim 14, wherein at least one of the catalytic molecule and reporter sequence is in a lyophilized form.
 19. The device according to claim 1, wherein the blood products comprise blood platelets.
 20. A method of testing a blood or blood product for a target molecule indicative of contamination in said blood or blood product, comprising providing a sample of a blood product in a compartment of the storage device for storing and testing blood or blood products, comprising: a container for receiving and storing blood or blood products; and at least one compartment for testing the blood or blood products, wherein said compartment comprises: at least a first section for holding a portion of the blood or blood products, and optionally testing the portion of the blood or blood products; contacting the blood product in the compartment with a lysing buffer; releasing the target molecule from the cells and protein in the blood product; and detecting the presence of the target molecule.
 21. The method according to claim 20, wherein the target molecule is a 16S ribosomal RNA or a nucleic acid associated with a pathogen.
 22. The method according to claim 20 or 21, wherein the detecting step employs test reagents comprising a catalytic molecule and a reporter sequence.
 23. The method according to claim 22, wherein said catalytic molecule is an inactivated ribozyme, a DNAzyme or a catalytic antibody.
 24. The method according to claim 22, wherein said test reagents are an inactivated ribozyme and an RNA reporter sequence.
 25. The method according to claim 24, wherein the inactivated ribozyme binds to the target molecule, which activates the ribozyme that cleaves the RNA reporter sequence and releases a detectable sequence.
 26. A method of testing a blood or blood product for a target molecule indicative of contamination in said blood or blood product, comprising providing a sample of a blood product in a compartment of the storage device for storing and testing blood or blood products, comprising: a container for receiving and storing blood or blood products; and at least one compartment for testing the blood or blood products, wherein said compartment comprises: at least a first section for holding a portion of the blood or blood products, and optionally testing the portion of the blood or blood products; contacting the blood product in the compartment with a buffer to dilute the sample; and detecting the presence of the target molecule.
 27. The method according to claim 26, wherein the target molecule is a protein associated with a pathogen.
 28. The method according to claim 26 or 27, wherein the detecting step employs test reagents comprising a catalytic molecule and a reporter sequence.
 29. The method according to claim 28, wherein said catalytic molecule is an inactivated ribozyme or catalytic antibody.
 30. The method according to claim 28, wherein said test reagents are an inactivated ribozyme and an RNA reporter sequence.
 31. The method according to claim 30, wherein the inactivated ribozyme binds to the target molecule which activates the ribozyme that cleaves the RNA reporter sequence and releases a detectable sequence. 